Ribonucleic acids (RNA) have central roles in all aspects of cellular metabolism. Protein synthesis is dependent upon the coordinated interactions of transfer RNA (tRNA), ribosomal RNA (rRNA), and messenger RNA (mRNA). The cellular roles of RNA are complex, as they function as structural elements (rRNA), catalytic components (in RNA processing), informational molecules (mRNA), and as a direct link between information contained in DNA and in proteins (tRNA). Gene activation is dependent not only on specific mRNA transcription but also on its correct splicing, capping and processing to create a functional mRNA. Consequently, in disease states such as cancer, where abnormal cell growth occurs, it is not unexpected that perturbations are indicative of metabolic alterations that can be exploited for cancer diagnosis or therapy.
RNA contains a variety of modified nucleosides, which are enzymatically modified post-transcriptionally by a variety of modification enzymes. Over 50 chemically distinct modifications have been described, each requiring a different enzyme. In humans and other animals normal RNA turnover and breakdown creates free modified nucleosides. These modified nucleosides collect in the circulation from which they are filtered and concentrated by the kidneys into the urine for excretion. Consequently, all urine contains levels of modified nucleosides that reflect RNA degradation in the organism.
Numerous reports have correlated increased rates of modified nucleoside urinary excretion with cancer and other disease states. Examples of such cancers include leukemias (Trweyn and Grever, 1986. CRC Crit. Rev. Clin. Lab. Sci., 24:71-93), chronic myelogenous leukemia (Heldman, et al. 1983. J. Lab. Clin. Med., 101:783-792), adult acute leukemia (Heldman, et al. 1983. Blood, 61:291-296), childhood acute lymphoblastic leukemia (Heldman, et al. 1983. J. Nat. Can. Inst., 71:269-273), bronchogenic carcinoma (Rasmuson, et al. 1983. Acta Radiolog. Oncology, 22:209-214), mammary and colorectal carcinoma (Rasmusen and Bjork, 1985. Bull. Mol. Biol. Med., 10:143-154), hepatocellular carcinoma (Tamura, 1986. Cancer, 57:1571-1575), small cell lung cancer (Tamura, 1986. Clin. Chem. Acta, 154:125-132) ovarian cancer (Oerlemans and Lange, 1986.), malignant lymphomas Rasmuson and Bjork, 1983.), nasopharyngeal carcinoma (Trewyn, 1982. Cancer 49: 2513-2517), and malignant mesothelioma (Fishbein, et al, 1983. Cancer Res. 43:2971-2974).
The monitoring of urinary modified nucleoside levels has not been demonstrated to be useful for cancer diagnosis, to stage disease, to determine the efficacy of a treatment a protocol or to monitor recurrence of cancers in remission. The present application provides one skilled in the art with the reagents and methodologies required to efficiently measure levels of modified nucleosides in a patient as a diagnostic or predictive assay useful in designing treatment protocols.